This Research Career Award application describes a mentored research program for the candidate, Dr. Mylonakis, in fungal pathogenesis. The candidate is trained in Infectious Diseases and proposes a research program to study virulence of Cryptococcus neoformans. The candidate's sponsor, Dr. Calderwood, has a Iongtime interest in using simple organisms as model hosts for the study of human pathogens, while the candidate's cosponsor, Dr. Heitman, has a Iongtime interest in the genetics of C. neoformans and has developed a number of related molecular biology protocols. The candidate has developed a novel system for studying genetic and molecular mechanisms of C. neoformans pathogenesis using the nematode model organism Caenorhabditis elegans. He has found that C. neoformans kills C. elegans, and several genes, such as those associated with signal transduction pathways, laccase production and the alpha mating type, previously shown to be involved in mammalian virulence, also play a role in C. elegans killing. He used this system to screen a bank of randominsertional mutants that were developed in the C. neoformans H99 background. From 350 mutants tested, 7 were identified as attenuated in C. elegans, with the hypovirulence persisting after the mutation was crossed to a clean genetic background. Genetic analysis of the first mutant revealed that the mutation occurred in a gene homologous to KIN1 of Saccharomyces cerevisiae. The SPECIFIC AIMS are as follows: 1) to develop and use the C. elegans system to screen a library of random C. neoformans insertional mutants and then to identify the disrupted genes and utilize a pathogenic wild type strain to make defined deletion mutations in each of the genes identified and develop the corresponding reconstituted strains, 2) to use the tail vein and lung inhalation murine models to test if the genes identified through the screen in C. elegans are involved in mammalian virulence, 3) to perform in-depth analysis for a limited number of selected mutants that demonstrate the most dramatic hypovirulence in both C. elegans and mice. The analysis will include evaluation of capsule, melanin production and mating. The research plan proposed with focus on the use of C. elegans as a facile model to study basic, evolutionarily conserved pathways associated with cryptococcal infection. Because C. neoformanshas similarities with other pathogenic yeasts, we expect that our findings will facilitate the study of fungus/host interaction in general.